The penetratin sequence in the anticancer PNC-28 peptide causes tumor cell necrosis rather than apoptosis of human pancreatic cancer cells.
BACKGROUND
PNC-27 and PNC-28 are p53-derived peptides from the human double minute (hdm-2) binding domain attached to penetratin. These peptides induce tumor cell necrosis of cancer cells, but not normal cells. The anticancer activity and mechanism of PNC-28 (p53 aa17-26-penetratin) was specifically studied against human pancreatic cancer.


METHODS
MiaPaCa-2 cells were treated with PNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH) and apoptosis as assayed for measuring elevation of proapoptotic proteins. PNC-29, an unrelated peptide, and hdm-2-binding domain p53 aa12-26 without penetratin (PNC-26) were used as controls. Since there is evidence that penetratin is required for tumor cell necrosis, we tested "naked" p53 peptide without penetratin by transfecting a plasmid that encodes p53 aa17-26 segment of PNC-28 into MiaPaCa-2 and an untransformed rat pancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy was employed to further elucidate anticancer mechanism.


RESULTS
Treatment with PNC-28 does not result in the elevation of proapoptotic proteins found in p53-induced apoptosis, but elicits rapid release of LDH, indicative of tumor cell necrosis. Accordingly, we observed membrane pore formation and dose-dependent killing. In direct contrast, transfected MiaPaCa-2 cells underwent apoptosis, and not necrosis, as evidenced by expression of high levels of caspases-3 and 7 and annexin V with background levels of LDH.


CONCLUSIONS
These results suggest that PNC-28 may be effective in treating human pancreatic cancer. The penetratin sequence appears to be responsible for the fundamental change in the mechanism of action, inducing rapid necrosis initiated by membrane pore formation. Cancer cell death by apoptosis was observed in the absence of penetratin.
Bowne
Wilbur B
WB
Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA. wilbur.bowne@va.gov
Sookraj
Kelley A
KA
Vishnevetsky
Michael
M
Adler
Victor
V
Sarafraz-Yazdi
Ehsan
E
Lou
Sunming
S
Koenke
Jesco
J
Shteyler
Vadim
V
Ikram
Kamran
K
Harding
Michael
M
Bluth
Martin H
MH
Ng
Mou
M
Brandt-Rauf
Paul W
PW
Hannan
Raqibul
R
Bradu
Stephan
S
Zenilman
Michael E
ME
Michl
Josef
J
Pincus
Matthew R
MR
D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D057846:Cell-Penetrating Peptides; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009336:Necrosis; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology*
C493956:CDKN1A protein, human; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D050759:Cyclin-Dependent Kinase Inhibitor p21; C513570:PNC-28; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; C414409:penetratin; D007770:L-Lactate Dehydrogenase; D051736:Proto-Oncogene Proteins c-mdm2; D020169:Caspases